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Journal of Bacteriology and Virology 2015. Vol. 45, No. 4 p.364 – 371 http://dx.doi.org/10.4167/jbv.2015.45.4.364 Communication

Detection of Bacterial Species in Tissues at Different Stages of Disease Severity

Da-Le Yoon1§, Shukho Kim2§, Hyesoon Song2, Yong-Gun Kim1, * * Jae-Mok Lee1 and Jungmin Kim2

1Department of , Kyungpook National University School of Dentistry, Daegu; 2Department of , Kyungpook National University School of Medicine, Daegu, Korea

The goal of this research was to determine the relationship between the stage of chronic periodontitis and the presence of six bacterial pathogens (Aggregatibacter actinomycetemcomitans: AA, : FN, : PG, intermedia: PI, faecalis: EF, and Parvimonas micra: PM). Forty-six chronic periodontitis patients visiting a dental hospital were included in this investigation. They were classified into four chronic periodontitis stages based on the sulcus bleeding index value and the probing depth. The tissue samples from the periodontal surgery were used for a direct PCR detection assay. A total of 49 samples from 46 patients were collected and classified into four chronic periodontitis groups (N: 6, P1: 13, P2: 18, P3: 12). The PCR assay showed that FN, PI, and PM were involved from the beginning of chronic periodontitis (P1), while AA and PG existed regardless of the disease stages. EF was strongly linked to the P3 stage of the disease. In order to assess the effect of dental treatments on patients with chronic periodontitis, EF should be a critical marker for P3 patients, while FN, PI, and PM would be good indicators for chronic periodontitis. Key Words: Chronic periodontitis, Oral , PCR detection

bacteria involved in , and various kinds INTRODUCTION of pathological toxic substances are secreted as disease progresses (2). can either avoid, weaken, Chronic periodontitis is an inflammatory disease that and/or neutralize host defense mechanisms and initiate results in the destruction of periodontal tissue, including the various immune pathological processes that can cause peri- alveolar bone and connective tissue that support the teeth odontal disease and even worsen disease condition. According (1). Periodontal tissue is capable of maintaining a healthy to Socransky et al., there are various inducing factors for status when there is a balance between the host defense and periodontitis, such as a decrease of bacteria in number that the toxicity of bacteria. However, there are many pathogenic play a protective role against pathogenic bacteria, local

Received: October 16, 2015/ Revised: October 27, 2015/ Accepted: October 30, 2015 § These authors equally contributed to this work. *Corresponding author: Jungmin Kim. Department of Microbiology, Kyungpook National University School of Medicine, Daegu 41944, Korea. Phone: +82-53-420-4845, Fax: +82-53-427-5664, e-mail: [email protected] *Corresponding author: Jae-Mok Lee. Department of Periodontology, Kyungpook National University School of Dentistry, Daegu 41940, Korea. Phone: +82-53-600-7522, Fax: +82-53-427-3263, e-mail: [email protected] **This research was supported by Kyungpook National University Research Fund (2012) and MEDIM foundation (2011). ○CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/license/by-nc/3.0/).

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Bacteria Detection from Chronic Periodontitis Samples 365 trauma, and the mental instability of the host (3, 4). These Fusobacterium nucleatum: FN, Porphyromonas gingivalis: factors promote the growth of bacteria that cause periodontal PG, Prevotella intermedia: PI, Enterococcus faecalis: EF, disease, and thereby allow them to overcome host defenses and Parvimonas micra: PM) in the chronic periodontitis so that periodontitis proceeds. Certain changes in the con- tissue samples from the patients with varying disease severity. dition for bacterial growth and in the host body, especially PCR is a highly sensitive detection tool that is widely used intraoral tissue, inhibit the host from effectively suppressing for the microbiological diagnosis of dental diseases. Many the growth of bacteria and inevitable tissue destruction that dental studies have been reported on oral bacteria-associated follows. Shift phenomenon of bacteria and periodic increase dental diseases and microbiomes of dental patients, which of bacteria which comes from the periodic weakening in used PCR and/or DNA sequencing (11, 12). We used a direct bacterial control incapability of the host bring the tissue PCR detection assay to detect the six pathogenic bacteria destruction. This phenomenon can be defined as cross- from tissue sample obtained from the periodontal surgery. reactivity between the defense factor and destructive factor, We also determined the relationship between the presence which causes periodic tissue destruction (5). As a result, oral of six pathogenic bacteria and the stages of chronic peri- bacteria irritate epithelial cells, initiating the periodontal odontitis. disease by triggering innate, inflammatory, and adaptive immune responses (6). Porphyromonas gingivalis, Prevotella MATERIALS AND METHODS intermedia, , Campylobacter rectus, Patients Eikenella corrodens, Fusobacterium nucleatum, Aggregati- bacter actinomycetemcomitans, Parvimonas micra, Tre- A total of 49 tissue samples were obtained from 46 ponema denticola, and Eubacterium are bacteria that are patients who visited Kyungpook National University Dental characteristically observed in chronic periodontitis. Speci- Hospital for chronic periodontal treatment. Accounting for fically, A. actinomycetemcomitans, C. rectus, P. gingivalis, the 49 samples, three patients each gave two chronic peri- P. intermedia, F. nucleatum, and T. forsythia are diversely odontitis samples that were taken from different regions of dispersed in the space with high periodontal disease reactivity the mouth with different severities of chronic periodontitis. (7, 8). In addition, in 2012, it was found that oral Entero- Informed consent was obtained, before the surgery, from the coccus faecalis possibly played a role as a reservoir for the all the patients enrolled in this study. The study protocol was transferable and antimicrobial resistance approved by the IRB of Kyungpook National University genes and was responsible for the pathogenesis of chronic Hospital (IRB Number: 74005-830). periodontitis (9, 10). Clinical measurement and sampling Therefore, it is critical to determine which bacterial species or strains are the major cause of chronic periodontitis. An The sulcus bleeding index (SBI) value and probing depth analysis of the quantities of periodontal pathogens, depending (PD) are the clinical periodontal parameters that can be on the severity or progression of chronic periodontitis, should obtained from six sites per tooth in an initial examination. follow for more accurate diagnosis and prevention of peri- Clinical criteria to describe gingiva were determined using odontitis progression. The identification of these pathogenic these parameters (13). The level of bone resorption could bacterial species will also help us set the main targets for be observed in the available radiographic images. Normal periodontitis therapies. However, specific pathogens that are and chronic periodontitis were classified according to the responsible for each stage in the progression of periodontal following criteria: normal (N) is clinically healthy gingiva disease are not yet fully known. without bleeding that shows no evidence of bone resorption In this study, we examined the presence of six patho- or periodontal pockets. Chronic periodontitis has more than genic bacteria (Aggregatibacter actinomycetemcomitans: AA, one periodontal pocket (≥ 5 mm) with at least one of them

366 D-L Yoon, et al.

Table 1. Primers used in this study

Primer name Primer sequence Target pathogen1 Amplicon size (bp) AA F3 5'-TGCGTAGAGATGTGGAGGAA-3' AA 165 AA B3 5'-GGCGGTCGATTTATCACGT-3' FN F3 5'-AGGCGATGATGGGTAGCC-3' FN 214 FN B3 5'-AGCCGTCACTTCTTCTGTTG-3' PG F3 5'-GGTAAGTCAGCGGTGAAACC-3' PG 218 PG B3 5'-GCGTGGACTACCAGGGTAT-3' PI F3 5'-ACGGCCTAATACCCGATGTT-3' PI 193 PI B3 5'-CTGCCTCCCGTAGGAGTT-3' EF16s-F 5'-CGCTTCTTTCCTCCCGAGT-3' EF 142 EF16s-R 5'-GCCATGCGGCATAAACTG-3' PM16s-F 5'-TCGAACGTGATTTTTGTGGAAA-3' PM 85 PM16s-R 5'-GGTAGGTTGCTCACGTGTTACTCA-3' 1AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra

showing ≥ 4 mm loss of attachment, a extracted from samples using a Genomic DNA Preparation bleeding index of 3, and/or clear evidence of bone resorp- Kit (HIGene Genomic DNA Prep Kit, BIOFACT, Korea) tion. Chronic periodontitis can be classified in more detail according to the manufacturer's instructions. PCR amplifi- according to its probe attachment level (PAL) as follow: If cation was performed in 50 μl of final solution containing the PAL is 1~2 mm, it is classified as P1 (mild chronic 5 μl of 10X PCR buffer (ExTaq, TaKaRa, Japan), 400 μM of periodontitis); if the PAL is 3~4 mm or ≥ 5 mm, it is each deoxynucleotide triphosphate (TaKaRa), 400 nM of classified as P2 or P3, respectively (14, 15). each primer, 1 U of Taq polymerase (ExTaq, TaKaRa), and Samples of subgingival tissue were collected from patients 2 μl of the extracted DNA samples. For the positive controls, with periodontal disease and from healthy individuals, at clinical isolates of Aggregatibacter actinomycetemcomitans the Department of Periodontology, School of Dentistry, (AA: KCOM 1299, 1304, 1306), Fusobacterium nucleatum Kyungpook National University. The samples were obtained (FN: KCOM 1276, 1322, 1323), Porphyromonas gingivalis from the patients during periodontal surgery, which included (PG: KCOM 2796, 2797, 2798), Prevotella intermedia (PI: surgical or tooth extraction by internal KCOM 1101, 1104, 1106), and Parvimonas micra (PM: bevel incision. Before the surgery, informed consent was KCOM 1037, 1533, 1535) were kindly provided by the provided to all of the study participants. Korean Collection for (KCOM, Korea). An ATCC (American Type Culture Collection) E. faecalis PCR detection (EF) strain 29212 was used for the positive PCR control. For the detection of six bacterial pathogens (A. actino- These positive control strains were harvested from growth mycetemcomitans, F. nucleatum, P. gingivalis, P. intermedia, media, prepared by boiling, and used for the PCR. Auto- E. faecalis, and P. micra), PCR primers targeting species- claved, distilled water was used for the negative PCR control. specific 16S rRNA coding regions of these pathogens were For the detection of AA, FN, PG, and PI, 35 PCR cycles chosen, synthesized, and used (16~18) (Table 1). DNA was of 30 sec at 95℃, 30 sec at 52℃, and 1 min at 72℃ were

Bacteria Detection from Chronic Periodontitis Samples 367

Table 2. PCR detection results of 49 samples including their clinical information

Sample Sampling data Chronic Sex1/Age AA3 FN PG PI EF PM number (yyyy.mm.dd) periodontitis2 1 2014.05.30 M/54 P2 ＋4 ＋ ＋ ＋ - ＋ 2 2014.04.02 F/45 P2 ＋ ＋ ＋ ＋ - ＋ 3 2014.03.18 M/52 P1 ＋ ＋ ＋ ＋ - ＋ 4 2014.04.21 F/53 N ＋ - ＋ - - - 5 2014.03.25 M/48 P2 ＋ ＋ ＋ ＋ - ＋ 6 2014.04.09 F/18 N ＋ ＋ ＋ - - - 7 2014.06.13 M/60 P2 ＋ ＋ ＋ - - ＋ 8 2014.06.13 F/57 P2 ＋ ＋ ＋ ＋ - ＋ 9 2014.05.16 M/58 P1 ＋ ＋ ＋ ＋ - - 10 2014.01.28 M/52 P2 ＋ ＋ ＋ ＋ - ＋ 11 2014.06.27 M/42 P2 ＋ ＋ ＋ - - - 12 2014.04.09 F/68 N ＋ ＋ ＋ - - ＋ 13 2014.04.03 M/35 P2 ＋ ＋ ＋ - - ＋ 14 2014.04.25 M/51 P2 ＋ ＋ ＋ ＋ ＋ ＋ 15 2014.04.02 F/59 P2 ＋ ＋ ＋ ＋ - ＋ 16 2014.03.07 M/39 N ＋ ＋ ＋ ＋ - ＋ 17 2014.06.16 M/54 P2 ＋ ＋ ＋ ＋ - ＋ 18 2014.02.24 F/42 P2 ＋ ＋ ＋ ＋ - ＋ 19 2014.04.16 M/53 N ------20 2014.05.09 F/48 N ＋ ＋ ＋ - - - 21 2014.02.07 M/60 P2 ＋ - ＋ - - - 22 2014.04.11 F/64 P2 ＋ ＋ ＋ ＋ - ＋ 23 2014.04.07 M/49 P1 ＋ ＋ ＋ - - - 24 2014.03.19 F/43 P2 ＋ - - - - - 25 2014.03.28 M/46 P3 ＋ ＋ ＋ ＋ ＋ ＋ 26 2014.06.16 F/53 P3 ＋ ＋ ＋ ＋ - ＋ 27 2014.04.11 F/51 P3 ＋ ＋ ＋ ＋ - ＋ 28 2014.04.11 F/35 P3 ＋ ＋ ＋ - - ＋ 29 2014.06.17 F/53 P3 ＋ ＋ ＋ ＋ ＋ ＋ 30 2014.04.07 F/59 P3 - - - - ＋ ＋ 31 2014.04.08 M/53 P3 ＋ ＋ ＋ ＋ ＋ ＋ 32 2014.06.20 M/45 P3 ＋ ＋ ＋ ＋ ＋ ＋ 33 2014.03.14 F/52 P3 + ＋ ＋ ＋ ＋ ＋ 34 2014.04.07 F/42 P3 ＋ ＋ ＋ ＋ ＋ ＋ 35 2014.04.02 F/55 P3 ＋ ＋ ＋ ＋ ＋ ＋ 36 2014.04.21 F/47 P3 ＋ ＋ ＋ ＋ ＋ ＋ 37 2014.05.09 M/49 P1 ＋ ＋ ＋ ＋ - ＋

368 D-L Yoon, et al.

Table 2. Continued

Sample Sampling data Chronic Sex1/Age AA3 FN PG PI EF PM number (yyyy.mm.dd) periodontitis2 38 2014.03.28 M/61 P1 ＋ ＋ ＋ ＋ - ＋ 39 2014.04.03 M/47 P1 ＋ ＋ ＋ ＋ - ＋ 40 2014.06.10 F/55 P1 ＋ ＋ ＋ - - ＋ 41 2014.04.30 F/46 P1 - ＋ ＋ ＋ - ＋ 42 2014.04.11 F/51 P1 ＋ ＋ ＋ ＋ - ＋ 43 2014.02.28 M/38 P1 ＋ ＋ ＋ ＋ - ＋ 44 2014.03.26 M/48 P2 ＋ ＋ ＋ - - ＋ 45 2014.06.09 M/54 P1 ＋ ＋ ＋ ＋ - ＋ 46 2014.04.28 F/48 P1 ＋ ＋ ＋ ＋ - ＋ 47 2014.04.02 M/37 P2 ＋ ＋ ＋ ＋ - ＋ 48 2014.04.08 M/48 P2 - ＋ ＋ ＋ - ＋ 49 2014.04.11 M/52 P1 ＋ ＋ ＋ ＋ - ＋ 1M: male, F: female 2Refer Materials & Methods for the abbreviation 3AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra 4＋: PCR positive, -: PCR negative.

carried out. For the detection of EF, 35 PCR cycles of 30 sec Statistical analysis at 95℃, 30 sec at 62℃, and 30 sec at 72℃ were performed. For the detection of PM, the latter PCR conditions, except The statistical analysis was performed using the Pearson's with an annealing temperature of 60℃, were accomplished. Chi-squared test in SPSS 12.0 (SPSS Inc., USA). An initial DNA denaturation step of 2 min at 95℃ was completed before the amplification cycles began, and for RESULTS & DISCUSSION each PCR reaction, the 35 cycles were followed by a final extension step of 7 min at 72℃. PCR was performed using The clinical data and characteristics of the recruited the TaKaRa PCR Thermal Cycler Dice (TaKaRa Bio). The patients and specimens were summarized in Table 2. Of PCR products were analyzed by 2% agarose gel electro- the 46 patients, 26 patients were male and 23 were female. phoresis containing RedSafeTM nucleic acid staining solution No participants were related to each other. Enrolled patient (Intron Biotech. Korea) in TRIS-borate-EDTA buffer with showed past medical history including diabetes mellitus a Mupid electrophoresis apparatus (Advance, Japan). The (10 patients), hypertension (8 patients), and hyperlipidemia DNA bands were visualized using a Gel Doc XR system (3 patients). The others had no specific systemic diseases. (Bio-Rad, USA). If the DNA band of the sample is visible The mean age of all patients was 49.5 yr with an 8.6 yr and corresponds to that of the positive PCR control, the standard deviation. The maximum age was 68 yr and the decision would be PCR positive. Otherwise would be judged minimum was 18 yr. There were no apparent differences in as PCR negative (non-matching or invisible). the mean ages between the groups (N: 46.50 yr, P1: 50.77 yr, P2: 49.94 yr, and P3: 49.25 yr).

Bacteria Detection from Chronic Periodontitis Samples 369

Table 3. Detection of six pathogenic bacteria from dental tissue samples by PCR

No. of PCR positive samples/No. of samples used for PCR (%) Sample groups classified by clinical measurement Bacteria1 P1, Mild chronic P2, Moderate chronic P3, Severe chronic N, Normal gingiva periodontitis periodontitis periodontitis (PAL2 ≤ 2 mm) (PAL ≤ 4 mm) (PAL > 4 mm) AA 5/6 (83.3) 12/13 (92.3) 17/18 (94.4) 11/12 (91.6) FN 4/6 (66.7) 13/13 (100) 15/18 (83.3) 11/12 (91.6) PG 5/6 (83.3) 13/13 (100) 16/18 (88.8) 11/12 (91.6) PI 1/6 (16.7) 11/13 (84.6) 12/18 (66.7) 10/12 (83.3) EF 0/6 (0) 0/13 (0) 1/18 (5.5) 9/12 (75.0) PM 2/6 (33.3) 12/13 (92.3) 15/18 (83.3) 12/12 (100) 1AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella intermedia, EF: Enterococcus faecalis, PM: Parvimonas micra 2PAL: probe attachment level.

According to PAL measurements, number of samples Table 4. Result of statistical analysis to determine the association classified into P1, P2, and P3 were 13, 18, and 12 samples, of six bacteria with the states of chronic periodontitis respectively. Six normal samples, from persons with no P value Bacteria1 evidence of bone resorption or periodontal pockets, were N versus P1 P1 versus P2 P2 versus P3 also evaluated in the study. Table 3 shows the frequencies AA 0.554 0.811 0.765 of detecting six bacterial species from 49 samples (normal, FN 0.028* 0.121 0.511 P1, P2, and P3 chronic periodontitis tissue). AA, FN, and PG 0.130 0.214 0.803 PG were the most frequently detected bacteria, regardless of PI 0.004* 0.259 0.311 a normal status or the level of chronic periodontitis. These EF - 0.387 0.0000772* bacteria were detected in more than 80% of all chronic PM 0.007* 0.462 0.136 periodontitis patients and more than 60% in healthy normal 1AA: Aggregatibacter actinomycetemcomitans, FN: Fusobacterium individuals included in the study. FN, PI and PM were highly nucleatum, PG: Porphyromonas gingivalis, PI: Prevotella inter- media, EF: Enterococcus faecalis, PM: Parvimonas micra prevalent in chronic periodontitis patients regardless of the *Statistical significance (p < 0.05). disease stage, but these bacteria were detected less frequently in normal samples (FN, PI and PM, 66.7%, 16.7% and 33.3%, respectively. p < 0.05). EF was not detected both in the normal and in P1 stage samples, but only one P2 sample tissues regardless of the disease stage. EF was strongly was PCR positive for EF (5.5%), unlike the other bacteria linked to the P3 stage in this study (Table 4). tested. However, 75% of P3 samples was PCR positive for AA, FN, and PG are well-studied bacterial pathogens in EF that was statistical significance relation between detection the field of chronic periodontitis pathogenesis. Fine et al. rate of P3 and those of the other samples (p < 0.01). Pearson's reported that AA is frequently associated with localized Chi-squared testing suggested that FN, PI, and PM were aggressive chronic periodontitis (LAP) and can serve as a associated with the beginning of chronic periodontitis (P1) risk marker for the initiation of LAP (19, 20). FN, on the while AA and PG were present in chronic periodontitis other hand, adheres to and invades human epithelial cells,

370 D-L Yoon, et al. thus, has been detected in subgingival plaque samples (21). periodontitis lesions are a more direct immunological source PG is the strongest bacterial marker for chronic periodontitis compared to subgingival plaque, using subgingival tissue and is highly associated with periodontal bone loss (22, 23). instead of plaque samples could be a more accurate and These three pathogens were detected in 83% of P1 to P3 reliable source for determining the impact of pathogens on patients, as well as 66.7~83.3% of normal participants in- host immunity. cluded in this study. These results indicate that these bacteria In summary, this is the first report on six chronic might be associated with early onset of chronic periodontitis periodontitis-associated pathogens and their association with in these patients, or may not play roles in the pathogenic stages of chronic periodontitis in Korea. Although the sample process of periodontitis. size of the study was small and target bacteria were restricted PI and PM were less prevalent in normal samples, but to only six microorganisms, our findings could suggest a occurred with high frequencies in P1 to P3 samples (p < direction for assessment of treatment of chronic periodontitis 0.01), suggesting that these two pathogens might be critically through the recognition of pathogens. Further analysis of associated with the disease, by triggering and/or worsening the quantities of periodontal, depending on the progression their chronic periodontitis. Interestingly, EF was detected of chronic periodontitis, should follow for more accurate with relatively high frequency (75%) only in P3 samples in diagnosis. In order to assess the effect of dental treatments this study. EF is a human commensal which lives in the on chronic periodontitis patients, EF would be a critical nutrient-rich, anaerobic, and ecologically variable environ- marker for P3 patients, while FN, PI, and PM would be good ment of the oral cavity and intestine (24, 25). Similar to our indicators for chronic periodontitis patients overall. results, Souto et al. reported that EF was detected more often in saliva and subgingival samples of chronic periodontitis REFERENCES patients than those of healthy individuals (26). EF harbors various virulence factors that may subsidize other bacteria 1) Pihlstrom BL, Michalowicz BS, Johnson NW. Peri- present in chronic periodontitis tissues (27). In addition, the odontal diseases. Lancet 2005;366:1809-20. deeper chronic periodontitis pockets in P3 patients have 2) Page RC, Offenbacher S, Schroeder HE, Seymour GJ, wider epithelial surface areas with preexisting dental bacteria Kornman KS. Advances in the pathogenesis of peri- where EF may attach easily. However, the potential odontitis: summary of developments, clinical impli- pathogenic role of EF in oral infections is still unknown due cations and future directions. Periodontol 2000;1997;14: 216-48. to limited number of study addressing this question. 3) Socransky SS, Haffajee AD. The Bacterial Etiology of According to a study performed in 2012 by Lee H et al. Destructive Periodontal Disease: Current Concepts. J (28), to analyze the change of bacterial flora based on the Periodontol 1992;63:322-31. stage of periodontitis, the relative levels of A. actinomy- 4) Socransky SS, Haffajee AD, Goodson JM, Lindhe J. cetemcomitans and C. rectus were positively correlated with New concepts of destructive periodontal disease. J Clin the progression of periodontal disease. In this study, large Periodontol 1984;11:21-32. amounts of EF, which was not a focus of the previous study, 5) Holt SC, Bramanti TE. Factors in virulence expression were detected in severe chronic periodontitis (P2, P3) samples, and their role in periodontal disease pathogenesis. Crit and it is equally important to note that EF was not detected Rev Oral Biol Med 1991;2:177-281. in healthy periodontal tissue. 6) Kinane DF, Demuth DR, Gorr SU, Hajishengallis GN, Unlike most previous studies, which used subgingival Martin MH. Human variability in innate immunity. plaque samples to evaluate the prevalance of periodontal Periodontol 2000;2007;45:14-34. pathogens (29), in this study, we identified bacterial pathogens 7) Socransky SS, Haffajee AD. Periodontal microbial from subgingival tissues. Since bacteria found in chronic ecology. Periodontol 2000;2005;38:135-87.

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